Lymphatic Vessel Imaging

描述（由申请人提供）：本R21项目的长期目标是开发活体小鼠淋巴管成像工具。淋巴管通过瓣膜状的开口排出间质液，它们运输脂质并参与免疫系统。它们将抗原和抗原呈递细胞运送到淋巴结，并将淋巴结的细胞运送到循环系统。当淋巴管被破坏，淋巴水肿，淋巴积聚在间质组织，发生。免疫功能受到阻碍。淋巴管生成发生在免疫部位的炎症和淋巴结（次级淋巴器官）中，在那里，lv和高内皮小静脉（HEVs）之间存在相互作用。淋巴管生成发生在异位部位的淋巴细胞积聚的慢性炎症中，称为三级淋巴组织（TLOs），具有淋巴结的许多特征。转基因大鼠胰岛素启动子（RIP）驱动淋巴毒素-1 （RIPLT1）的小鼠在胰腺中表现出这样的TLOs。已经用于培养可通过绿色荧光蛋白成像的hev小鼠的技术将用于培养可通过红色荧光蛋白类似成像的lv小鼠。为实现这一目标，将实现以下具体目标：目的：培养淋巴管表达荧光蛋白的转基因小鼠。pCLASPER重组技术将用于分离LV基因（prox1、lyve-1或podoplanin），插入红色荧光报告基因，如tdTomato，并产生转基因小鼠。2. 评价淋巴报告基因小鼠的表达保真度。我们将分析经淋巴管报告基因构建的转基因小鼠在稳定状态下的几种组织、免疫后的淋巴结和RIPLT1小鼠的TLOs中内源性LV基因和转基因基因的共表达。3. 目的：评价活体小鼠的lv。多光子体内显微镜将用于评价荧光lv。免疫小鼠淋巴结中（绿色）高内皮小静脉和（红色）淋巴管之间的相互作用将被实时分析。对淋巴管生成和左室功能的动态理解将为淋巴功能和淋巴水肿功能障碍的治疗提供见解。淋巴管成像工具的发展将有助于了解在免疫反应中lv和hev是如何被调节、维持和合作的，并有助于了解炎症、移植、自身免疫、传染病和癌症中的淋巴管生成。

DESCRIPTION (provided by applicant): The long term goal of this R21 project is to develop tools for imaging lymphatic vessels (LVs) in living mice. Lymphatic vessels drain interstitial fluid by means of valve-like openings, they transport lipids and they participate in the immune system. They transport antigen and antigen presenting cells to lymph nodes and cells from the lymph node to the circulation. When lymphatic vessels are disrupted, lymphedema, the accumulation of lymph in the interstitial tissues, occurs. Immunological functions are thwarted. Lymphangiogenesis occurs in inflammation at the site of immunization and in lymph nodes (secondary lymphoid organs) where there is an interaction between LVs and high endothelial venules (HEVs). Lymphangiogenesis occurs in chronic inflammation in lymphoid accumulations in ectopic sites, called tertiary lymphoid tissues (TLOs) that have many characteristics of lymph nodes. Mice transgenic for the rat insulin promoter (RIP) driving lymphotoxin-1 (RIPLT1) exhibit such TLOs in the pancreas. Techniques that have been used to develop mice whose HEVs can be imaged via green fluorescent protein will be used here to develop mice whose LVs can be similarly imaged via a red fluorescent protein. To attain this goal, the following specific aims will be accomplished: 1. To develop transgenic mice whose lymphatic vessels express fluorescent proteins. The pCLASPER recombineering technology will be used to isolate LV genes (prox1, lyve-1, or podoplanin), insert red fluorescent reporter genes, such as tdTomato, and produce transgenic mice. 2. To evaluate lymphatic reporter transgenic mice for fidelity of expression. Mice transgenic for lymphatic vessel reporter constructs will be analyzed for co- expression of endogenous LV genes and transgenes in several tissues in the steady state, in lymph nodes after immunization, and in TLOs in RIPLT1 mice. 3. To evaluate LVs in living mice. Multiphoton in vivo microscopy will be used to evaluate fluorescent LVs. Interactions between (green) high endothelial venules and (red) lymphatic vessels in the lymph nodes of immunized mice will be analyzed in real time. A dynamic understanding of lymphangiogenesis and LV function will provide therapeutic insight into lymphatic function and dysfunction in lymphedema. The development of imaging tools for lymphatic vessels will provide insight into how LVs and HEVs are regulated, maintained, and cooperate during an immune response and provide insight into lymphangiogenesis in inflammation, transplantation, autoimmunity, infectious diseases, and cancer. PUBLIC HEALTH RELEVANCE: A thorough understanding of lymphangiogenesis and lymphatic vessel function will provide therapeutic insight into lymphatic function and dysfunction in conditions such as lymphedema. Imaging tools for lymphatic vessels will provide insight into how this vascular system is maintained and regulated during an immune response and provide insight into lymphangiogenesis in inflammation, transplantation, autoimmunity, infectious diseases, and cancer. )